Table 2.

Summary of PLA conditions employed on human brain sections. The conditions shown (antigen retrieval procedure, blocking solution, primary antibody dilutions, PLA reaction amplification time) were employed on 2–4 control and 2–4 iPD human brain sections (striatum and substantia nigra) for each condition

Antigen retrieval	Blocking solution	1° Ab dilutions	Amplification time
Citrate buffer (pH 6.0)	Donkey Serum	1:500 each	100 min, 37°C
high temperature (95°C)	1 h, RT
Citrate buffer (pH 6.0)	Donkey Serum	1:500 each	100 min, 37°C
high pressure	1 h, RT
Citrate buffer (pH 6.0)	Donkey Serum	1:500 each	130 min, 37°C
high temperature (95°C)	1 h, RT
Citrate buffer (pH 6.0)	Donkey Serum	1:500 each	130 min, 37°C
high pressure	1 h, RT
Citrate buffer (pH 6.0)	Donkey Serum	1:200 each	130 min, 37°C
high pressure	1 h, RT
Citrate buffer (pH 6.0)	Duolink Blocking	1:200 each	130 min, 37°C
high pressure	30 min, 37°C
Citrate buffer (pH 7.0)	Donkey Serum	1:100 each	130 min, 37°C
high pressure	1 h, RT
Citrate buffer (pH 6.0)	Donkey Serum	1:100 each	130 min, 37°C
high pressure	1 h, RT
Citrate buffer (pH 6.0)	Donkey Serum	1:50 each	130 min, 37°C
high pressure	1 h, RT
–	Donkey Serum	1:500 each	130 min, 37°C
	1 h, RT

Parallel PLA assays were performed on inducible HEK cells overexpressing WT and G2019S LRRK2 to assure that the assay per se was working. The penultimate condition is described in detail in the Materials and Methods section. The last condition was exactly as described by Keeney et al. (2021) [23]. Representative images for the last two conditions are shown in Fig. 9.